Development of a continuous microbial fermentation system using reduced genome E. coli that undergo programmed autolysis and generate nuclease post-production for the manufacture of biopharmaceuticals

Continuous bioprocessing that brings with it the advantages of lower ultimate cost, a smaller equipment footprint and energy efficiency, is beginning to be embraced by the biopharmaceutical industry. Long term continuous fermentation requires a high level of genetic and metabolic stability. Scarab Genomics proprietary reduced genome E. coli strains provide the requisite genetic and metabolic stability for such long term continuous fermentations. Scarab Genomics proposes to develop the first fully continuous platform for the production and purification of recombinant proteins and plasmid DNA (pDNA) by replacing homogenization of bacteria with a custom autolysis system controlled by a temperature sensitive promoter. Distinctive features of Scarab’s Clean Genome bacteria such as the absence of all cryptic prophage make it uniquely suited for tightly regulated lysis. Having attained proof-of-concept in small scale culture, this application proposes to optimize this lysis system in high density (>200 OD600) continuous fermentation (C-Flow). To further facilitate the development of a continuous system, the lysis operon will also drive the exonuclease bensonase so that as lysis occurs, the large amount of nucleic acid that hinders downstream processing will be removed. To enable a lysis system for plasmid DNA (pDNA) purification, bensonase in the lysis operon will be replaced by the lambda exonuclease which digests genomic DNA but not pDNA thereby providing an alternate system for the production of pDNA. Once optimized, this lysis and nucleic acid digestion system will be used to express two commercially relevant test proteins. The first will be insulin because Scarab sees the possibility of C-Flow as a vehicle for the production of less expensive insulin and insulin related compounds to meet the objectives of the US government and the WHO. The second will be a single chain antibody (scFv) given the numerous applications in diagnostics and therapeutics for single chain antibodies and antibody fragments. To prepare C-Flow for market, the application also proposes to develop upgrades to the current system including oxygen infusion of medium using microbubble delivery and a microbubble separation chamber for the removal of cell debris. These innovations will result in the first continuous fermentation platform in E. coli for the production of biopharmaceuticals.